This project involves a genetic and biochemical study of the regulation of gene expression controlling the metabolism of galactose in the unicellular eukaryote Tetrahymena. We have found that transcription of the galactokinase gene is repressed by high concentrations of glucose and by the hormone epinephrine acting through increased intracellular levels of cyclic AMP (Roberts and Morse, P.N.A.S. 75:1810, 1978). We are now analyzing the mechanisms by which glucose may modulate intracellular levels of epinephrine and, thereby, cyclic AMP, as well as the possible involvement of a cyclic AMP-dependent protein kinase in galactokinase gene regulation. Experimental analyses are conducted in parrallel with wild-type cells and otherwise isogenic mutant strains which we have selected for potential alterations in glucose metabolism, the beta-adrenergic system, protein kinase, and the galactokinase structural gene and its regulation. In addition, the construction of a Tetrahymena gene-bank using a lambda vector system, and the screening of this bank for galactokinase gene sequences (using as probe either antibody to Tetrahymena galactokinase, or galactokinase mRNA) will provide material with which we can quantitate galactokinase-specific mRNA levels in vivo by RNA-DNA hybridization. These studies should provide insights into the fundamental mechanisms of hormone and cyclic nucleotide-dependent regulation of gene expression in eukaryotic cells.